Limits...
Dimerization of lipocalin allergens.

Niemi MH, Rytkönen-Nissinen M, Miettinen I, Jänis J, Virtanen T, Rouvinen J - Sci Rep (2015)

Bottom Line: We have determined two different dimeric crystal structures for bovine dander lipocalin Bos d 2, which was earlier described as a monomeric allergen.The crystal structure analysis of all other determined lipocalin allergens also revealed oligomeric structures which broadly utilize inherent structural features of the β-sheet in dimer formation.According to the moderate size of monomer-monomer interfaces, most of these dimers would be transient in solution.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biocenter Kuopio, University of Eastern Finland, PO BOX 111, 80101 Joensuu, Finland.

ABSTRACT
Lipocalins are one of the most important groups of inhalant animal allergens. The analysis of structural features of these proteins is important to get insights into their allergenicity. We have determined two different dimeric crystal structures for bovine dander lipocalin Bos d 2, which was earlier described as a monomeric allergen. The crystal structure analysis of all other determined lipocalin allergens also revealed oligomeric structures which broadly utilize inherent structural features of the β-sheet in dimer formation. According to the moderate size of monomer-monomer interfaces, most of these dimers would be transient in solution. Native mass spectrometry was employed to characterize quantitatively transient dimerization of two lipocalin allergens, Bos d 2 and Bos d 5, in solution.

No MeSH data available.


Native mass spectra of lipocalin allergens at different protein concentrations.(a,b) rBos d 2 at protein (monomer) concentrations of 10 and 90 μM. The peaks representing the protein monomer (M) are in blue, while the peaks representing the protein dimer (D) are in red. The most abundant peaks for the monomer and the dimer have been assigned with the number indicating the ion charge state (i.e., M7+ = [rBos d 2 + 7H]7+). (c-h) Native mass spectra of Bos d 5 at a protein (monomer) concentration of 1-25 μM.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4561914&req=5

f4: Native mass spectra of lipocalin allergens at different protein concentrations.(a,b) rBos d 2 at protein (monomer) concentrations of 10 and 90 μM. The peaks representing the protein monomer (M) are in blue, while the peaks representing the protein dimer (D) are in red. The most abundant peaks for the monomer and the dimer have been assigned with the number indicating the ion charge state (i.e., M7+ = [rBos d 2 + 7H]7+). (c-h) Native mass spectra of Bos d 5 at a protein (monomer) concentration of 1-25 μM.

Mentions: Native MS was used to study the dimer formation of rBos d 2 in native-like solution conditions (i.e., 10 mM ammonium acetate, pH 6.9). The basic protocol for native MS was similar to our previous work3, which allows for the weak transient protein dimers to be detected. The native MS spectrum of rBos d 2 (10 μM in respect to the protein monomer) at pH 7 showed signals of both monomers and dimers in solution (Fig. 4a). When the protein concentration was increased to 90 μM, the relative intensity of the dimer increased accordingly (Fig. 4b). These results indicate that rBos d 2 is indeed a weak transient dimer in solution. To further quantify the dimerization (see Methods for details), we obtained protein monomer–dimer ratios from the native mass spectra over a wide range of protein (monomer) concentrations (0–90 μM). The fitted curve of the free monomer concentration against the total protein concentration is presented in Fig. 5a. The determined value for the equilibrium dissociation constant (KD) for self-association was KD = 340 ± 20 μM. We also analyzed the pH-dependence of the dimerization (Fig. 5c), and the amount of rBos d 2 dimer never exceeded the amount of the monomer. The amount of dimer slowly decreased when the pH changed to be more alkaline. The maximal amount of dimer was present at around pH 3.


Dimerization of lipocalin allergens.

Niemi MH, Rytkönen-Nissinen M, Miettinen I, Jänis J, Virtanen T, Rouvinen J - Sci Rep (2015)

Native mass spectra of lipocalin allergens at different protein concentrations.(a,b) rBos d 2 at protein (monomer) concentrations of 10 and 90 μM. The peaks representing the protein monomer (M) are in blue, while the peaks representing the protein dimer (D) are in red. The most abundant peaks for the monomer and the dimer have been assigned with the number indicating the ion charge state (i.e., M7+ = [rBos d 2 + 7H]7+). (c-h) Native mass spectra of Bos d 5 at a protein (monomer) concentration of 1-25 μM.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4561914&req=5

f4: Native mass spectra of lipocalin allergens at different protein concentrations.(a,b) rBos d 2 at protein (monomer) concentrations of 10 and 90 μM. The peaks representing the protein monomer (M) are in blue, while the peaks representing the protein dimer (D) are in red. The most abundant peaks for the monomer and the dimer have been assigned with the number indicating the ion charge state (i.e., M7+ = [rBos d 2 + 7H]7+). (c-h) Native mass spectra of Bos d 5 at a protein (monomer) concentration of 1-25 μM.
Mentions: Native MS was used to study the dimer formation of rBos d 2 in native-like solution conditions (i.e., 10 mM ammonium acetate, pH 6.9). The basic protocol for native MS was similar to our previous work3, which allows for the weak transient protein dimers to be detected. The native MS spectrum of rBos d 2 (10 μM in respect to the protein monomer) at pH 7 showed signals of both monomers and dimers in solution (Fig. 4a). When the protein concentration was increased to 90 μM, the relative intensity of the dimer increased accordingly (Fig. 4b). These results indicate that rBos d 2 is indeed a weak transient dimer in solution. To further quantify the dimerization (see Methods for details), we obtained protein monomer–dimer ratios from the native mass spectra over a wide range of protein (monomer) concentrations (0–90 μM). The fitted curve of the free monomer concentration against the total protein concentration is presented in Fig. 5a. The determined value for the equilibrium dissociation constant (KD) for self-association was KD = 340 ± 20 μM. We also analyzed the pH-dependence of the dimerization (Fig. 5c), and the amount of rBos d 2 dimer never exceeded the amount of the monomer. The amount of dimer slowly decreased when the pH changed to be more alkaline. The maximal amount of dimer was present at around pH 3.

Bottom Line: We have determined two different dimeric crystal structures for bovine dander lipocalin Bos d 2, which was earlier described as a monomeric allergen.The crystal structure analysis of all other determined lipocalin allergens also revealed oligomeric structures which broadly utilize inherent structural features of the β-sheet in dimer formation.According to the moderate size of monomer-monomer interfaces, most of these dimers would be transient in solution.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biocenter Kuopio, University of Eastern Finland, PO BOX 111, 80101 Joensuu, Finland.

ABSTRACT
Lipocalins are one of the most important groups of inhalant animal allergens. The analysis of structural features of these proteins is important to get insights into their allergenicity. We have determined two different dimeric crystal structures for bovine dander lipocalin Bos d 2, which was earlier described as a monomeric allergen. The crystal structure analysis of all other determined lipocalin allergens also revealed oligomeric structures which broadly utilize inherent structural features of the β-sheet in dimer formation. According to the moderate size of monomer-monomer interfaces, most of these dimers would be transient in solution. Native mass spectrometry was employed to characterize quantitatively transient dimerization of two lipocalin allergens, Bos d 2 and Bos d 5, in solution.

No MeSH data available.